The terpenoids, also called isoprenoids, constitute the largest family of natural products with over 22,000 individual compounds of this class having been described. The triterpenes or terpenoids (hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, polyprenols, and the like) play diverse functional roles in plants as hormones, photosynthetic pigments, electron carriers, mediators of polysaccharide assembly, and structural components of membranes. The majority of plant terpenoids are found in resins, latex, waxes, and oils.
Triterpenoids are of relevance to a variety of plant characteristics, including palatability to animals, and resistance to pathogens and predators. Triterpenes are mostly stored in plant roots as their glycosides, saponins (see Price K. R. et al, 1987, CRC Crit. Rev. Food Sci. Nutr. 26:27-133). Thus, for example, mutants of the diploid oat species, Avena strigosa, which lack the major oat root saponin, avenacin A-1 (so called saponin-deficient or “Sad” mutants) have been shown to have compromised disease resistance (Papadopoulou K. et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:12923-12928). These mutants have increased susceptibility to a number of different root-infecting fungi, including Gaeumannomyces graminis var. tritici, which is normally non-pathogenic to oats. Genetic analysis suggests that increased disease susceptibility and reduced avenacin content are causally related. Furthermore, a sad mutant which produces reduced avenacin levels (around 15% of that of the wild type) gives only limited disease symptoms when inoculated with G. graminis var. tritici in comparison to other mutants which lack avenacins completely, providing a further link between avenacin content and disease resistance.
There is an accumulating amount of data suggesting that saponins in the diet may be beneficial (see for example Shi, J. A. et al. (2004) J. Med. Food 7:67-78 and V is, E. H. et al. (2005) Nutr. Cancer 51:37-44). Similarly, dietary saponins of soybean have been shown to be beneficial in preventing hypercholesterolemia and aortic atherosclerosis in rats (Oakenfull, et al. (1984) Nutr. Rep. Int. 29:1039-1046). Since saponins are carried over from the bean into soy isolate with only minimal loss, increased levels of saponins in beans should lead to increased amounts of saponins in isolate (Berhow, M. A. et al. (2002) Phytochem. Anal. 13:343-348; Hu J., et al. (2002) J. Agric. Food Chem. 50:2587-2594). Increasing levels of saponins in beans, thus, would be an effective way of increasing saponin amounts in the human diet. In addition, the increase in saponins could provide a source for compounds used in drug development.
Triterpenes, as well as sterols, are synthesized via the isoprenoid pathway. In this pathway, two molecules of farnesyl pyrophosphate are joined head-to-head to form squalene, a triterpene. Squalene is then converted to
2,3-oxidosqualene. Various oxidosqualene cyclases catalyze the cyclization of 2,3-oxidosqualene to form various polycyclic skeletons, including one or more of cycloartenol, lanosterol, lupeol, isomultiflorenol, β-amyrin, α-amyrin, and thalianol.
This cyclization event catalyzed by oxidosqualene cyclases forms a branch point between the sterol and triterpene saponin biosynthetic pathways. The various oxidosqualene cyclases are evolutionarily related (Kushiro, T., et al. (1998) Eur. J. Biochem. 256:238-244) and produce a wide variety of three-, four-, and five-ring structures that can be further modified.
Triterpenoid saponins are synthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give pentacyclic triterpenoids, primarily oleanane (β-amyrin) or dammarane skeletons. The triterpenoid backbone then undergoes various modifications (oxidation, substitution, and glycosylation), mediated by cytochrome P450-dependent monooxygenases, glycosyltransferases (GTs), and other enzymes. In general very little is known about the enzymes and biochemical pathways involved in saponin biosynthesis. The genetic machinery required for the elaboration of this important family of plant secondary metabolites is as yet largely uncharacterized, despite the considerable commercial interest in this important group of natural products. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies. However, the first dedicated step in saponin biosynthesis is now understood to be carried out by the oxidosqualene cyclase β-amyrin synthase (the product of the Sad1 gene), which has recently been cloned and characterized (Haralampidis K. et al., 2001, Proc. Natl. Acad. Sci. U.S.A. 98:13431-13436). Another key step, mediated by the cytochrome P450 enzyme AsCYP51H10 (encoded by the Sad2 gene), has also recently been studied (Qi X. et al., 2006, Proc. Natl. Acad. Sci. U.S.A 103:18848-18853). AsCYP51H10 (SAD2) is required for avenacin synthesis. The precise biochemical function of AsCYP51H10 is not known. However Sad2 mutants accumulate β-amyrin and so AsCYP51H10 is likely to be required for oxidation of β-amyrin (or a derivative of this) at one or more positions (C12, C13, C16, C21 and/or C30) (FIG. 1).
Structural comparisons (FIG. 1) predict that other classes of enzyme in addition to cytochrome P450s will also be required for conversion of β-amyrin to avenacin A-1. These include glycosyltransferases (GTs), acyl transferases, and methyl transferases (MTs). Glycosyltransferases belong to a large family of enzymes that transfer saccharide units from activated donor molecules onto a wide spectrum of potential acceptor molecules. The array of potential acceptors includes proteins, lipids, polysaccharides and small molecules, which may be involved in diverse cellular processes such as cell wall synthesis and signalling (Coutinho P M et al., 2003, J. Mol. Biol., 328:307-317). Of seventy-seven GT families with representatives spanning all Kingdoms, the GT Family 1 is one of the largest (Coutinho P M and Henrissat B, 1999: Carbohydrate active enzymes website http://afmb.cnrs-mrs.fr/CAZY/). Family 1 consists of GTs that operate via an inverting catalytic mechanism of sugar transfer, usually onto low molecular weight acceptor molecules (Vogt T and Jones P, 2000, Trends Plant Sci. 5:380-386; Lim E-K and Bowles D J, 2004, EMBO J. 23:2915-2922). The branched sugar chain of avenacin A-1 is predicted, by analogy to other glycosylated small molecules, to be synthesized by the sequential addition of sugar units onto the aglycone component, most probably by the activity of three different glycosyltransferases (GTs). The first step in glycosylation involves the addition of L-arabinose onto the C3 hydroxyl group of the aglycone, mediated by an arabinosyltransferase. This is followed by the addition of two D-glucose molecules, one at the C2 position of the arabinose and the other at the C4 position, mediated by one (or possibly two) glucosyltransferases (Townsend B et al., 2006, Phytochemistry Revs. 5:109-114).
Acylation is a common feature of plant-derived natural products and alters their chemical and physical properties. It is therefore likely to influence the biological effects of natural products in ecological interactions and to influence other key processes such as subcellular trafficking and sequestration (for example by serving as a vacuolar uptake or retention tag). A new class of plant acyltransferases has recently been discovered. These enzymes—serine carboxypeptidase-like acyl tranferases—share homology with peptidases but lack peptidase activity and instead are able to acylate natural products (Milkowski C & Strack D (2003) Phytochemistry 65: 517; Fraser C M et al. (2005) Plant Physiology 138:1136). While other plant acyltransferases commonly use coenzyme thioesters as acyl donors these SCPLs use acyl glucose donors. The best-characterized members of the SCPL acyltransferase family are the tomato enzyme GAC-Lp, which catalyses the formation of glucose polyesters that contribute to insect resistance in wild tomato (Li A X & Steffens J C, 2000, PNAS 97: 6902); the Arabidopsis enzyme SNG1, which is required for the synthesis of the phenylpropanoid sinapoylmalate (a UV protectant) (Landry L G et al., 1995, Plant Physiology 109:1159; Lehfeldt C et al., 2000, Plant Cell 12:1295); a second Arabidopsis enzyme SNG2, which is involved in synthesis of sinapoyl choline in the seeds (Shirley A M et al., 2001, Plant J 28:83); and the Brassica napus acyltransferase BnSCT, which catalyses the formation of sinapate esters associated with bitterness, astringency and seed oil extraction problems (Milkowski C et al., 2004, Plant J. 38:80; Baumert A et al., 2005, Phytochemistry 66:1334). Many other important plant-derived natural products are known from biochemical analysis to be produced by glucose-ester-dependent acyltransferase reactions although the enzymes and genes involved in these modifications have not been characterized. Examples include the antioxidant chlorogenic acid in sweet potato (Ipomoea batatas), anthocyanins in wild carrot (Daucus carota), gallotannins in oak (Quercus robur) and sinapoyl- and benzoyl-esterified glucosinolates in brassicas (Milkowski C & Strack D (2003) Phytochemistry 65: 517; Fraser C M et al. (2005) Plant Physiology 138:1136). There are four different structurally related avenacins [14]. Avenacins A-1 (the major avenacin found in oat roots) and B-1 are acylated with N-methyl anthranilic acid, and avenacins A-2 and B-2 with benzoic acid (Hostettmann K and Marston A, 1995, Saponins, Cambridge University Press, Cambridge, UK).
S-Adenosyl-L-methionine-dependent methyltransferases are involved in O-methylation of many plant natural products (Frick S. et al. 2001, Phytochemistry 56:1-4). These enzymes play important roles in the synthesis of lignin precursors and other compounds required for plant defense (Gang D R et al 2002, Plant Cell 14:505-519.